Diagnosis and Monitoring of Renal Failure Using Peptide Biomarkers

ABSTRACT

Methods for the determination of renal failure, especially chronic renal failure and acute kidney injury, by measurement of peptide or protein biomarkers are described. The methods are useful to determine stages of renal failure, especially the early stages such as stage 1, 2, and 3 of chronic renal failure and stages R and I of acute kidney injury. Furthermore there are described peptides and test kits used in the invention. The described methods are intended to replace or complement the measurement of creatinine and/or cystatin C and/or NGAL for diagnosis of renal failure.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/544,605, filed Aug. 20, 2009, which claims the benefit of priority toU.S. Provisional Application No. 61/090,853, filed Aug. 21, 2008, all ofwhich are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The application relates to biomarkers and methods for the diagnosis ofrenal failure. The invention targets to diagnose all stages of chronicrenal failure, including early stages which are difficult to determinewith commonly used diagnostic methods known in the art. The biomarkersand methods of the invention are also used for the diagnosis of acuterenal failure or acute kidney injury. The advantage of the invention isthat the kidney function can be described accurately and therefore renalfailure can be detected at early stages and tracked through all stagesof progression.

BACKGROUND OF THE INVENTION

Renal failure is distinguished between acute renal failure or acutekidney injury and chronic renal failure. The basic difference betweenacute and chronic renal failure is the progression of the disease, whichis fast for acute renal failure (usually within days or weeks) and slowfor chronic renal failure (usually in the range of years). Both acuterenal failure and chronic renal failure can result in a complete loss ofrenal function, causing the subject to depend on renal replacementtherapy, such as hemodialysis or kidney transplantation. The incidenceof renal failure has doubled since 1990, most likely caused by theincrease of the incidence of diabetes and hypertension, the two majorcauses for kidney failure.

Chronic renal failure is an irreversible destruction of renal functionand usually is associated with the decreased size of the kidneys. Itdoes not cause pain, which is an important reason that it is often notdiagnosed until late stages, reducing the success rate of therapy.Treatment usually is started when the kidneys are already largely andirreversibly destroyed. In contrast, acute renal failure can bereversible, depending on its cause and on the success of the therapy.The success of the therapy depends, to a large extent, on the earlydiagnosis.

Renal failure can be determined by calculating the glomerular filtrationrate (GFR). The GFR describes the total volume of primary urine which isfiltered by both kidneys within a distinct time interval. The GFR isnormally >90 ml/min/body surface. It can be calculated by measuringeither exogenic markers or endogenic markers.

Specifically, the GFR can be determined by injecting known amounts ofexogenic diagnostic substances such as Inulin, radioactive substances(such as Chromium (Cr51-EDTA), Technetium (99mTC-DTPA), and Cobalt(Co57-vitamin B12)), or radiopaque material (such as Iohexol andIothalamate), and subsequently measuring the excretion of thesesubstances.

However, these tests are time-consuming, inconvenient, and expensive asthey require collecting and measuring the blood and/or the urineexcreted repeatedly over a specified time period. Other disadvantagesare the use of potentially harmful materials, such as radioactive orradiopaque materials, and the use of expensive and difficult to measuresubstances, such as Inulin. Thus, calculating the GFR using exogenicsubstances is usually only used in scientific studies and not clinicalpractice.

Renal failure can also be determined using endogenic biomarkers, amongwhich serum creatinine clearance is the most commonly used in clinicalpractice. Creatinine originates from muscle tissue and is increasinglysecreted by renal tubules with decreasing renal function. Creatinineclearance can be used to diagnose chronic, as well as acute, renalfailure.

Serum creatinine levels depend on age, sex, diet, muscle mass, ethnicbackground, physical activity, and disease. It is also secreted by thetubuli of the kidneys independent of kidney disease (Nephrol DialTransplant, 2002, 17 (Suppl. 7):7-15) or can be secreted via the gut.All these factors impair the reliability of creatinine clearance fordiagnosis of renal failure.

Various correction formulas are known to improve the accuracy ofcreatinine-based GFR determinations, by including additional metadatasuch as race, weight, body height, and body surface. However, theseformulas may not work correctly under certain circumstances. Forexample, if the subjects are bodybuilders, vegetarians, obese, or sufferfrom diabetes, hypertension, or chronic liver disease. In addition,these formulas are not useful in determining the GFR of healthy subjectsor of subjects suffering from only mild or moderate renal failure.Finally, the GFR may decline as much as 50% before serum creatininestarts to increase above reference levels. These limitations demonstratethat correction formulas often are not suitable for all stages of renalfailure (from mild GFR reduction to renal failure).

Another endogenic biomarker for renal failure diagnosis is Cystatin C, a120 amino acid, 20 kDa cysteine-protease inhibitor present in most bodyfluids. It is excreted by the kidneys. In contrast to creatinine, theexpression of Cystatin C is relatively constant, independent of, e.g.,muscle mass, body weight and age. Consequently Cystatin C is used as abiomarker of chronic renal failure. Some newer studies also indicatethat it might also be useful as a diagnostic marker for acute renalfailure (Kidney Int'l., 2004, 66:1115-1122). However, this has to befurther investigated.

Cystatin C can be determined directly from blood samples. Therefore,there is no need for the time-consuming, inconvenient and error proneurine collection. Nevertheless, there are still limitations for timelyand sensitive diagnosis of renal failure using Cystatin C. In addition,the suitability of Cystatin C for diagnosis of acute renal failure hasnot yet been validated.

NGAL is another promising endogeneous marker that appears to detectearly stages of acute renal injury. NGAL has been shown to correlatewith acute kidney injury in a variety of disease modalities and isdetectable at an early stage of disease. The validity of NGAL fordiagnosis of acute renal failure has yet to be fully determined.

Early detection and intervention is critical to effective treatment ofeffective treatment. However, the existing diagnosis methods have manydisadvantages and limitations as described. Therefore, a need exists fornovel methods for early diagnosis of renal failure.

BRIEF SUMMARY OF THE INVENTION

The present invention provides biomarkers, methods, and test kits fordiagnosis of renal failure. In one aspect, the renal failure is chronicrenal failure. In another aspect, the renal failure is acute renalfailure.

The present invention provides a peptide biomarker having the sequenceof SEQ ID NO:1 or 2. The present invention also provides a nucleic acidencoding a peptide biomarker having the sequence shown in SEQ ID NO:1 or2. The present invention further provides an antibody that specificallybinds to a peptide biomarker having the sequence shown in SEQ ID NO:1 or2. Also disclosed is a nucleic acid molecule, or a recombinant nucleicacid molecule consisting of a polynucleotide encoding the biomarker.

The present invention provides a method of identifying a subject havingor at risk of developing a renal disorder. The method comprisesobtaining a test biological sample from the subject, and determining thequantity of a peptide biomarker in the test biological sample. In oneembodiment, the biomarker is selected from the group consisting of SEQID NOs: 1 and 2.

In one aspect, the method further comprises the steps of obtaining acontrol biological sample from a control subject not having and not atrisk of developing a renal disorder, determining the quantity of thepeptide biomarker in the control sample, and comparing the quantity ofthe biomarker in the test sample and in the control sample. A varianceof the quantity of the biomarker in the test sample relative to thecontrol sample indicates that the subject has or is at risk ofdeveloping the renal disorder.

In another aspect, the method comprises comparing the quantity of thebiomarker with a predetermined reference value. A variance of thequantity of the biomarker in the test sample relative to the referencevalue indicates that the subject has or is at risk of developing a renaldisorder.

The present invention also provides a method of monitoring theprogression or regression of a renal disorder in a subject in needthereof, the method comprises obtaining a test biological sample from asubject and determining the quantity of a peptide biomarker in the testbiological sample over a period of time. The quantity of the biomarkercorrelates to a stage of the renal disorder. A difference of thequantities of the biomarker over a period of time is indicative of theprogression or regression of the renal disorder. In one embodiment, thebiomarker is selected from the group consisting of SEQ ID NOs: 1 and 2.

The present invention further provides a method of monitoring theefficacy of a renal disorder therapy in a subject in need thereof, themethod comprises obtaining a test biological sample from a subject, anddetermining the quantity of the peptide biomarker in the test biologicalsample prior to and subsequent to the therapy. The quantity of thepeptide biomarker obtained prior to therapy is compared to the quantityof the peptide biomarker obtained subsequent to therapy. The differenceis indicative of the efficacy of the renal disorder therapy. In oneembodiment, the renal disorder therapy is the administration ofmedication. In another embodiment, the renal disorder therapy ishemodialysis. In yet another embodiment, the renal disorder therapy iskidney transplantation. The biomarker is selected from the groupconsisting of SEQ ID NOs: 1 and 2, and the quantity of the biomarkercorrelates to a stage of the renal disorder.

The present invention further provides a method of determining therenal-toxic side effects of substances (such as medications, chemicals,and microbial or other toxins), living organisms (such as plant, animal,and microbes), and environmental influences (such as food ingestion,alcohol ingestion, consumption of tobacco products, stress, and physicalactivity), on a human or an animal. A first test biological sample isobtained from the subject prior to the event, and a second testbiological sample from the subject is obtained after the event. Thequantities of a peptide biomarker in the first test sample and thesecond test sample are determined. The quantity of the biomarkercorrelates to a stage of the renal disorder. The difference between thequantities is indicative of the effect of the event on renal function.

In certain embodiments of all the aspects, the subject is selected fromthe group consisting of a human, a primate, a mouse, a dog, a pig, and arat. The methods of the present invention can be used to determine thestages of renal failure. In one aspect, the methods of the presentinvention can be used to determine stages 1, 2, 3, 4, and 5 of chronicrenal failure. In another aspect, the methods of the present inventioncan be used to determine stages Normal, R, I, F, L, and E of acute renalfailure.

In certain embodiments of all the aspects, the quantity of the biomarkeris determined by calculating glomerular filtration rate. The biologicalsample is selected from the group consisting of blood, plasma, serum,hemofiltrate, urine, kidney tissue, in vitro cultured kidney cell lines,in vitro cultured primary kidney cells, in vitro cultured kidney tissue,in vitro cultured kidney organ, and supernatants from in vitro cellculture, tissue culture, and organ culture.

Certain embodiments of the aspects may further include a method ofinjecting the subject with an exogenic substance and calculatingglomerular filtration rate by measuring the clearance of the exogenicsubstance. The exogenic substance can be Inulin, a radioactive substance(e.g., Cr51-EDTA, 99mTC-DTPA, and Co57-vitamin B12), or a radiopaquematerial (e.g., Diatrizoates, Iodipamide, Iohexol, Iopamidol, Ioversol,Iothalamate, Ioxaglate, and Metrizamide). The glomerular filtration ratecan be calculated by measuring the clearance of an endogenic substance.The endogenic substance can be selected from the group consisting ofserum creatinine, Cystatin C, NGAL, urea, interleukin 18, intestinalform of alkaline phosphatease, N-acetyl-beta-gulcosaminidase,alanine-aminopeptidase, kidney injury molecule 1, parathyroid hormone,creatol, creatine kinase, methylguanidine, and 1,5-anhydroglucitol(1,5-AG).

In certain embodiments, the subject has a condition selected from thegroup consisting of injury to a kidney, type 2 diabetes, hypertension,cardiovascular disease, sepsis, hemorrhage, massive blood loss,congestive heart failure, decompensated liver cirrhosis, damaged kidneyblood vessels, obstructions of urine collection systems or extra-renaldrainage, vasculitis, malignant hypertension, acute glomerulonephritis,acute interstitial nephritis, and acute tubular necrosis. Further, incertain embodiments, the event is selected from the group consisting ofexposure to a substance, exposure to a living organism, food ingestion,alcohol ingestion, consumption of tobacco products, exposure to stress,and physical activity. The substance can be selected from the groupconsisting of a medication, a chemical, and a toxin. The living organismcan be selected from the group consisting of a plant, an animal, amicrobe, and a virus.

In certain embodiments, the size of the kidneys is determined, urineoutput is measured, urine sediments are analyzed, and excretion ofsodium or urea is analyzed. Kidney size can be determined using animaging technique, e.g., ultrasound.

In yet another aspect, the step of determining the quantity of thebiomarker is accomplished by an immunological method (e.g., an ELISAassay, an RIA assay, an ELI-Spot assay, a flow cytometry assay, animmunohistochemistry assay, a Western blot analysis, and a protein chipassay), a molecular biologic method (e.g., PCR analysis, a RT-PCRanalysis, a TaqMan PCR analysis, a nucleic acid chip assay, in situhybridization, a nucleic acid dot blot analysis, a nucleic acid slotblot analysis, a Southern blot analysis or a Northern blot analysis), ora physical method (e.g., mass spectrometric method, a FRET assay, achromatographic assay, or a dye-detection assay). The mass spectrometricmethod can be a MALDI, ESI, ionspray, thermospray, MCI, FAB, SELDI,ICAT, iTRAQ, or affinity mass spectrometric method. In certain aspects,the step of determining the quantity of the biomarker can beaccomplished by nuclear magnetic resonance (NMR), fluorometry,colorimetry, radiometry, luminometry, liquid chromatography, capillarychromatography, thin-layer chromatography, plasmon-resonance (e.g.BIACORE), or one- or two-dimensional gel electrophoresis.

The biological sample can be fractionated prior to the detection step.Fractionating can be done through a method selected from the groupconsisting of a chromatography method, a filtration method, a capillaryelectrophoresis method, a gel electrophoresis method, a liquidextraction method, a precipitation method, and an immunoprecipitationmethod. Further, the chromatography method can be reverse phasechromatography.

The methods of the present invention can be combined with knowndiagnostic methods using markers or procedures such as creatinineclearance, Cystatin C, or other markers for renal failure, or withmeasuring urine output, measuring the size of the kidneys, analysis ofurine sediments and excretion of sodium or urea to further improve theoverall sensitivity and/or specificity of the diagnosis of renalfailure.

Another aspect of the invention is the pre-analysis of a diagnosticsample intended for other non-renal failure diagnostic procedures. Thepre-analysis determines whether renal failure is present, as renalfailure might impair other markers for non-renal failure diseases orconditions, which are intended to be measured using the same sample.

The present invention further provides a test kit for diagnosing,monitoring the progression or regression, or monitoring the treatment ofa renal disorder. In one aspect, the kit comprises a peptide biomarker,a nucleic acid molecule consisting of a polynucleotide encoding apeptide biomarker, or a recombinant nucleic acid molecule consisting ofa polynucleotide encoding a peptide biomarker having the sequence of SEQID NO:1 or 2. In yet another aspect, the kit comprises an antibodyspecifically binds to a peptide biomarker having the sequence shown inSEQ ID NO:1 or 2. In one embodiment, the kit of the present inventionmay optionally comprise instructions for diagnosing, monitoring theprogression or regression, or monitoring the treatment of the renaldisorder. In certain aspects, instructions to detect the biomarker inthe biological sample are included in the kit.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts Peptide Displays of different stages of chronic renalfailure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides biomarkers, methods, and test kits fordiagnosis of renal failure. In one aspect, the renal failure is chronicrenal failure. In another aspect, the renal failure is acute renalfailure. “Chronic renal failure” is used interchangeably with chronicrenal disease, and “acute renal failure” is used interchangeably foracute renal disease, and/or acute kidney injury. Renal failure isregarded as chronic if it persists for at least 3 months.

The stages (alternatively termed states, classes or grades) of chronicrenal failure are defined by the GFR as stated in Table 1 (KidneyInt'l., 2005, 67:2089-2100).

TABLE 1 Stages of chronic renal failure Stage GFR (mL/min) Description 1≧90 Normal or elevated GFR 2 60-89 Mild GFR reduction 3 30-59 ModerateGFR reduction 4 15-29 Severe GFR reduction 5  <15 Renal failure

Acute renal failure is classified into 5 distinct stages using the“RIFLE” (Risk, Injury, Failure, Loss, End-stage renal disease) stagingsystem as shown in Table 2 (The Lancet, 2005, 365:417-430). The first 3stages are distinguished based on both increased serum creatinine levelsand reduced urine output.

TABLE 2 Stages of acute renal failure GFR (based on Stage serumcreatinine) criteria Urine output criteria Risk Serum creatinineincreased 1.5 times <0.5 mL/kg/h for 6 h Injury Serum creatinineincreased 2.0 times <0.5 mL/kg/h for 12 h Failure Serum creatinineincreased 3.0 times, <0.3 mL/kg/h for 24 h, or creatinine >355 mM/L whenthere or anuria for 12 h was an acute rise of >44 mM/L Loss Persistentacute renal failure — >4 weeks End-stage End-stage renal disease >3months —

Other staging methods for renal failure resulting in similar orcomparable classifications of different stages of renal failure may beused according to the invention.

Determining the presence or absence of renal failure means to determineif renal failure, including chronic renal failure or acute renalfailure, is present or is not present in a subject. This includes thedetermination of any stage of the chronic renal failure, e.g., stages1-5 of the chronic renal failure shown in Table 1. It also includes thedetermination of any stage of the acute renal failure, e.g., stagesRIFLE of the acute renal failure shown in Table 2. It further includesdistinguishing the stages of renal failure, e.g., distinguishing stage 1from stages 2 and/or 3, or distinguishing stages 1, 2, or 3 from stages4 and/or 5.

Samples and Subjects

Any sample containing peptides and/or proteins from a subject to betested for the presence or absence of renal failure can be usedaccording to the invention. The sample can be whole blood, plasma,serum, hemofiltrate, urine, kidney or other tissue, or a fractionthereof or a combination of the samples. The sample may also originatefrom in vitro cultured cell lines, or primary cells, or from in vitrocultured tissue or organs, especially kidneys, or from cell culture,tissue culture, or organ culture supernatants.

The sample may be pooled samples collected during a certain period oftime or of more than one subject. It can be used directly or afterstorage for various time periods at various storage conditions. Thesample may or may not be pre-processed, e.g., fractionated bychromatography, filtration, capillary electrophoresis, precipitation,liquid or other extraction methods, and immunoprecipitation.

The subject from which the sample originates can be a human or an animalused for experimental testing, preferably a mammal, such as a primate, adog, a pig, or a rodent, including, e.g., a mouse or a rat.

Specifically, subjects having vascular diseases, including bilateralrenal artery stenosis, ischemic nephropathy, hemolytic-uremic syndrome,and vasculitis, are more likely to develop chronic renal failure. Othercauses of chronic renal failure include, but not limited to, focalsegmental glomerulosclerosis, IgA nephritis, diabetic nephropathy, lupusnephritis, polycystic kidney disease, drug and toxin-induced chronictubulointerstitial nephritis, kidney stones, and prostate diseases.

Causes of acute renal failure are divided into 3 main groups: pre-renal,post-renal and intra-renal. Pre-renal causes include lack of sufficientblood supply of the kidneys, which in turn may be caused by hemorrhage,massive blood loss, congestive heart failure, decompensated livercirrhosis (liver cirrhosis with complications such as bleedings,ascites), damaged kidney blood vessels, sepsis, or systemic inflammationdue to infection. Post-renal causes include obstructions of urinecollection systems or extra-renal drainage, which in turn may be causedby medication interfering with normal bladder emptying, prostatediseases, kidney stones, abdominal malignancy (such as ovarian cancer orcolorectal cancer), or obstructed urinary catheter. Intra-renal causesare renal tissue-destroying effects, such as vasculitis, malignanthypertension, acute glomerulonephritis, acute interstitial nephritis andacute tubular necrosis. They can be caused by, e.g., ischaemic events(such as hemoglobinuria, myoglobinuria, and myoloma) or by nephrotoxicsubstances (such as antibiotics, radio contrast agents, uric acid, andoxalate). Subjects suffering from these diseases and conditions are morelikely to develop acute renal failure.

Biomarkers as Diagnostics for Renal Failure

A biomarker is an organic biomolecule present in a sample used todetermine the phenotypic status of the subject or predict aphysiological outcome (e.g., health or disease state). A biomarker isdifferentially present between different phenotypic statuses if the meanor median expression level of the biomarker in the different groups iscalculated to be statistically significant. A single biomarker, or acombination of particular biomarkers, provides measures of relative riskor probability that a subject belongs to one phenotypic status oranother or will develop membership to a specific phenotypic status.Therefore, they are useful as biomarkers for disease (diagnostics),therapeutic effectiveness of a drug (theranostics), drug toxicity, andpredicting and identifying the immune response.

Biomarkers according to the invention include proteins, proteinfragments, peptides, and nucleic acids.

Peptides and Proteins

In one aspect, the present invention provides a peptide biomarker havingthe sequence of AGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGP (SEQ ID NO:1). Thispeptide is a fragment of the full length protein Collagen alpha-1(III)(P02461). The present invention also provides a peptide biomarker havingthe sequence of ELEPPEQQEPGERQEPS (SEQ ID NO:2). This peptide is afragment of the full length protein Integrin alpha-7 (Q13683).

The term “peptide” refers to compounds consisting of two or more aminoacids joined covalently by peptide bonds having a molecular mass of 0.5to 20 kDa. The term “protein” refers to a polypeptide bigger than apeptide.

A full length protein refers to the complete sequence of a protein withthe signal sequence. For example, full length human cystatin C(NP_(—)000090) has 146 amino acid residues. Fragments of full lengthproteins are considered peptides, including signal sequences and othersequences created during proteolytic or other processing, fragments suchas pro-peptide sequences, as long as the fragments have at least 7 aminoacid residues and can be identified as originating form the parentprotein sequence by use of standard sequence alignment algorithms asknown in the art, such as the BLAST or FASTA algorithms. For example,amino acids 1-26 of the full length human cystatin C (NP_(—)000090) is asignal peptide.

The terms “peptide” and “protein” according to the invention includecompounds containing only amino acids, as well as compounds alsocontaining non-amino acid constituents such as carbohydrates and lipidstructures and include compounds containing only peptide bonds as wellas compounds containing other bonds, e.g. ester, thioether or disulfide.

The peptides of the present invention can be fragments of proteinspresent in nature. Peptides and proteins may contain postranslationalmodifications such as phosphate groups, carbohydrate groups or lipidmoieties. Also included are peptides and proteins that comprise aminoacids different from the standard set of 20 amino acids coded by thegenetic code.

Modifications of peptides or proteins according to the invention maycomprise modifications due to postranslational modifications, chemicalmodifications, enzymatic modifications and modifications due to othermechanisms. Examples of possible modifications include but are notlimited to: glycosylation, phosphorylation, sulphatation, pyroglutamatemodification, cystein-disulfide bridges, methylation, acetylation,acylation, farnesylation, formylation, geranylgeranylation,biotinylation, stearoylation, palmitylation, lipolyation,C-mannosylation, miristoyliation, amidation, deamidation, methylation,demethylation, carboxylation, hydroxylation, iodination, oxidation,pegylation, prenylation, ADP-ribosylation, addition of lipids, ofphosphatidylinositol, of glycosylphosphatidylinositol (GPI)-anchor, ofpyridoxal phosphate, modification of cystein residues resulting incarboxyamidomethylcysteine, resulting in carboxymethylcysteine, orresulting in pyridylethylcysteine, modification of lysine residuesresulting in liponic acid, modification of glutamic acid resulting inpyroglutamic acid.

Modifications of peptides or proteins according to the invention maycomprise unusual amino acids, chemically or enzymatically modified aminoacids including, but not limited to: alpha amino butyric acid, betaamino butyric acid, beta amino iso-butyric acid, beta alanine, gammabutyric acid, alpha amino adipic acid, 4-amino benzoic acid, amino ethylcysteine, alpha amino penicillanic acid, allysine, 4-carboxy glutamicacid, cystathionine, carboxy glutamic acid, carboxy amido methylcysteine, carboxy methyl cysteine, cystein acid, citrulline,dehydroalanine, di-amino butyric acid, dehydro amino-2-butyric acid,ethionine, glycine-proline di-peptide, 4-hydroxyproline, hydroxylysine,hydroxyproline, homoserine, homo cysteine, histamine, iso-valeine,lysinoalanine, lanthionine, norvaline, norleucine, ornithine,2-pipiridine-carboxylic acid, pyroglutamic acid, pyrrolysine,proline-hydroxy proline di-peptide, sarcosine, 4-selenocysteine,syndesine, and thioproline. Further examples can be found in databasessuch as “Delta Mass” athttp://www.abrf.org/index.cfm/dm.home?AvgMass=all.

Nucleic Acids, Complementary and Degenerated Nucleic Acids andDerivatives

In another aspect, the present invention provides a nucleic acidencoding the peptide biomarker having sequence of SEQ ID NO:1 or 2. Thepresent invention also provides a recombinant nucleic acid containingthe nucleic acid sequence encoding the peptide biomarker of SEQ ID NO:1or 2.

Nucleic acids are deoxyribonucleic acids (DNA), ribonucleic acids (RNA),or combinations thereof, regardless if the nucleic acid molecules aresingle or double-stranded, linear, circular or branched. It will beunderstood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C).

The nucleic acid molecules are purified nucleic acid molecules, i.e.,the preparation contains at least 50%, and preferably higher, of acertain nucleic acid molecule or of a certain mixture of more than onecertain nucleic acid molecule. Examples of nucleic acid molecules aregenomic DNA, cDNA (complementary DNA), mRNA (messenger RNA),recombinantly produced nucleic acid molecules corresponding to thenatural sequences, chemically synthesized nucleic acid moleculescorresponding to the natural sequences, or nucleic acid moleculesgenerated by enzymatic methods (such as by use of polymerase chainreaction (PCR)), or nucleic acid molecules purified from natural sources(such as prokaryotic or eukaryotic cells, blood cells, body fluids suchas plasma, serum, urine, biopsy material (especially kidney biopsymaterial), using methods known in the art.

Furthermore, nucleic acid molecules according to the invention can bepartial or complete derivatives of nucleic acids, including nucleotidesor nucleotide-like molecules not found in nature (such asphosphorothioates, peptide nucleic acids (PNAs), N3′,P5′-phosphoramidates, morpholino phosphoroamidates, 2′-O-methoxyethylnucleic acids, 2′-fluoro-nucleic acids, arabino-nucleic acids, andlocked nucleic acids (LNA, ribonucleotides containing a methylene bridgethat connects the 2′-oxygen of ribose with the 4′-carbon)).

Antibodies

In another aspect, the present invention provides an antibody thatspecifically binds to the peptide biomarker having the sequence of SEQID NO:1 or 2. Such an antibody may be polyclonal or monoclonal. Theantibodies of the present invention are useful in the methods and testkits of the invention. For example, after an antibody is provided, apeptide marker can be detected and/or quantified using any of a numberof well recognized immunological binding assays.

One skilled in the art would readily carry out known techniques in orderto raise antibodies against the biomarkers of the invention. See, e.g.,Coligan, Current Protocols in Immunology (1991); Harlow & Lane,Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies:Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature256:495-497 (1975). Such techniques include, but are not limited to,antibody preparation by selection of antibodies from libraries ofrecombinant antibodies in phage or similar vectors, as well aspreparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246: 1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

Determining a Biomarker

Determining a biomarker includes the qualitative and/or quantitativedetermination and measurement of biomarkers that are present inbiological samples. Depending on the sensitivity of the measurementmethod used, the determination may detect the presence of a substance,i.e., the quantity of the substance is above the detection limit of themethod used, or it may be that the substance is not detectable, i.e.,its quantity is below the detection limit of the method used.

If a quantitative determination is performed, the quantity of thesubstance may be determined as a relative or as an absolute value,depending on the method used. If the determination is absolute, it maybe expressed as an exact quantity of the biomarker (e.g., in mg/mL, mmolor M). If the determination is relative, it may be relative to areference measurement, to a reference or normal value, or to a referencesample. The relative quantity may be expressed, e.g., as a percentage ofthe reference or in arbitrary units.

Generally, all methods suitable to detect and analyze biomarkers in asample can be used in the methods of the invention. These methodsinclude physical, immunological, and molecular biology methods.

Suitable physical methods include mass spectrometric methods,fluorescence resonance energy transfer (FRET) assays, chromatographicassays (such as liquid chromatography, capillary chromatography, andthin-layer chromatography), and dye-detection assays. Mass spectrometricmethods include, but are not limited to, matrix-assisted laserdesorption ionisation (MALDI), continuous or pulsed electrosprayionization (ESI), and related methods such as ionspray or thermospray ormassive cluster impact (MCI). The ion sources can be matched withdetection formats including linear or non-linear reflectiontime-of-flight (TOF), single or multiple quadrupole, single or multiplemagnetic sector, fourier transform ion cyclotron resonance (FTICR), iontrap, and combinations thereof, e.g. ion-trap/time-of-flight. Forionization, numerous matrix/wavelength combinations (MALDI) or solventcombinations (ESI) can be used. Other suitable mass spectrometricmethods are, e.g., fast atom bombardment (FAB) mass spectrometry,Surface Enhanced Laser Desorption/Ionization (SELDI) mass spectrometry,isotope coded affinity tag (ICAT) mass spectrometry, iTRAQ massspectrometry, and affinity mass spectrometric methods.

Suitable immunologic methods include, but are not limited to, enzymelinked immuno assays (ELISA), sandwich, direct, indirect, or competitiveELISA assays, enzyme-linked immunospot assays (ELISPOT), radio immunoassays (RIA), flow cytometry assays (FACS=fluorescence activated cellsorting), immunohistochemistry, Western blot, protein-chip assays usingfor example antibodies, antibody fragments, receptors, ligands, or otheragents binding the substances, such as the peptides of the presentinvention. Binding agents can be used to analyze and determine thepresence or absence of biomarkers in a sample. The biological sample iscontacted with one or more agents that bind to the biomarker todetermine the level of expression of the biomarker in the sample. One ormore of the agents may be operably linked to a detectable label.

Molecular biology methods that can be used according to the inventioninclude, but are not limited to, Northern or Southern blothybridization, nucleic acid dot- or slot-blot hybridization, in situhybridization, nucleic acid chip assays, PCR, reverse transcriptase PCR(RT-PCR), or real time PCR (taq-man PCR).

Other methods to detect biomarkers include, e.g., nuclear magneticresonance (NMR), fluorometry, colorimetry, radiometry, luminometry, orother spectrometric methods, plasmon-resonance (e.g. BIACORE), and one-or two-dimensional gel electrophoresis.

Furthermore, different methods may be combined to determine a biomarker.For example, the combination of liquid chromatography and massspectrometry (LC-MS) or the combination of capillary zoneelectrophoresis and mass spectrometry (CE-MS), or the combination ofextraction of certain classes of substances such as peptides, from asample prior to LC-MS or prior to CE-MS or prior to other methods.

Combination of Methods

The methods of the present invention can be combined with otherdiagnostic methods for renal failure to improve specificity andselectivity. For example, other diagnostic methods include measurementsof exogenic markers, e.g., Inulin, radioactive materials (such asCr51-EDTA, 99mTC-DTPA, and Co57-vitamin B12), and radiopaque materials(such as Diatrizoates, Iodipamide, Iohexol, Iopamidol, Ioversol,Iothalamate, Ioxaglate, and Metrizamide).

Other diagnostic methods also include measurements of endogenic markers,such as serum creatinine and Cystatin C. Additional biomarkers for renalfailure have been described in the literature. For example, biomarkersfor acute renal failure include NGAL, urinary interleukin 18, tubularenzymes (such as the intestinal form of alkaline phosphatase,N-acetyl-beta-gulcosaminidase and alanine-aminopeptidase), and kidneyinjury molecule 1. Some biomarkers, such as parathyroid hormone(parathormone), a peptide hormone, may be used to distinguish acute fromchronic renal failure (Ren. Fail., 2007, 29:509-512). In addition, knownmodified forms of biomarkers for renal failure, such as creatininemodified by hydroxyl radical addition resulting in Creatol (Ren. Fail.,2007, 29:279-283), or enzymes modifying creatine, such as creatinekinase may also be suitable for diagnosis of renal failure. Examples ofnon-peptide markers for renal failure include methylguanidine (used incombination with creatol), and 1,5-anhydroglucitol (1,5-AG) (used incombination with serum creatinine) (Nephron, 1996, 73:707-709).

Further diagnostic methods include imaging techniques to determine thesize of the kidneys (such as ultrasound), measurement of urine volume,analysis of urine sediments, and analysis of urinary chemistry (such asexcretion of sodium or urea).

It is possible to combine one or more than one of these diagnosticmethods with the method of the invention. In addition, the diagnosticprocedures can be done simultaneously or successively to confirm thediagnosis or to increase the sensitivity and/or specificity of thediagnosis.

Use of the Methods and Biomarkers of the Invention

The methods and biomarkers of the invention can be used, among others,for diagnosis of renal failure, including chronic and acute renalfailure. The methods and biomarkers of the invention can be used fordetermining the stages of renal failure, especially early stages ofrenal failure which are difficult or not at all possible to diagnoseusing methods known in the art.

The present invention provides a method of identifying a subject havingor at risk of developing a renal disorder, the method comprisesobtaining a test biological sample from the subject and determining thequantity of a peptide biomarker in the test sample. In one embodiment,the biomarker is selected from the group consisting of SEQ ID NOs: 1 and2.

In one aspect, the method further comprises the steps of obtaining acontrol biological sample from a control subject not having and not atrisk of developing a renal disorder, and comparing the quantity of thebiomarker in the test sample and in the control sample. A variance ofthe quantity of the biomarker in the test sample relative to the controlsample indicates that the subject has or is at risk of developing therenal disorder.

In another aspect, the method further comprises comparing the quantityof the biomarker with a predetermined reference value. A variance of thequantity of the biomarker in the test sample relative to the referencevalue indicates that the subject has or is at risk of developing a renaldisorder.

The methods and biomarkers of the invention can also be used todetermine the stages of renal failure. In one aspect, the methods andbiomarkers of the invention can be used to distinguish chronic renalfailure stages 1, 2, 3, 4 and 5 from each other. In another aspect, themethods and biomarkers of the invention can be used to distinguish acuterenal failure stages R, I, F, L and E from Normal and from each other.

Peptide Displays from different stages of chronic renal failure areshown in FIG. 1. To generate Peptide Displays, samples are prepared andseparated by means of RP-HPLC (reversed phase high pressure liquidchromatography) and peptides eluting from the HPLC column are collectedinto 96 fractions. Each fraction is subjected to MALDI-TOF-MS(matrix-assisted laser desorption/ionization time-of-flight massspectrometry) and the mass spectra of all 96 fractions are combined,resulting in an in silico two-dimensional display of peptide masses,where the x-axis displays the mass-to-charge ratio, the y-axis isdetermined by the retention time on the RP-HPLC or the fraction numberand signal intensity is depicted by color saturation. FIG. 1demonstrates the accumulation of peptides in blood plasma of subjects.

The present invention also provides a method of monitoring theprogression or regression of a renal disorder in a subject in needthereof, the method comprises obtaining a test biological sample fromthe subject; determining the quantity of a peptide biomarker in the testsample; repeating the steps over a period of time; and comparing thequantity of the biomarker over the period of time. In one embodiment,the biomarker is selected from the group consisting of SEQ ID NOs: 1 and2.

The present invention further provides a method of monitoring theefficacy of a renal disorder therapy in a subject in need thereof, themethod comprises determining the quantity of a peptide biomarker in atest biological sample obtained from the subject prior to and subsequentto the therapy. In one embodiment, the renal disorder therapy is theadministration of medication. In another embodiment, the renal disordertherapy is hemodialysis. In yet another embodiment, the renal disordertherapy is kidney transplantation.

The present invention further provides a method of determining therenal-toxic side effects of substances (such as medications, chemicals,and microbial or other toxins), living organisms (such as plant, animal,and microbes), and environmental influences (such as food ingestion,alcohol ingestion, consumption of tobacco products, stress, and physicalactivity), on a human or an animal.

The methods and biomarkers of the invention can also be used to pre-testa sample to determine if the subject from which the sample originatessuffers from kidney failure and for this reason shows an alteredexcretion profile of other biomarkers intended to be measured fordiagnostic purposes. After the methods and biomarkers of the inventionhave confirmed that the subject from which the sample originates, doesnot suffer from kidney failure, additional diagnostic biomarkers can bedetermined, without being in doubt that a possibly underlying kidneyfailure impairs measurement of the additional diagnostic biomarker.Alternatively the pre-test for kidney failure and the test for theadditional biomarker can be done simultaneously, or the results of thepre-test for kidney failure can be converted into a correction factorused to correct the result of the renal-failure-impaired measurement ofthe additional biomarker, based on the extent to renal failure, or basedon correction factors determined for individual biomarkers by empiricaltesting.

Test Kits

The present invention further provides a test kit for diagnosing,monitoring the progression or regression, or monitoring the treatment ofa renal disorder. In one embodiment, the kit comprises a peptidebiomarker having the sequence of SEQ ID NO:1 or 2. In anotherembodiment, the kit comprises a nucleic acid encoding a peptidebiomarker having the sequence shown in SEQ ID NO:1 or 2. In yet anotherembodiment, the kit comprises an antibody that specifically binds to apeptide biomarker having the sequence shown in SEQ ID NO:1 or 2.

The test kit may optionally comprise substances for use as standardand/or controls. The test kit may further optionally comprise knownmarkers for renal failure, such as creatinine or Cystatin C, to furtherincrease overall sensitivity and/or specificity of the test.

In one aspect, the present invention provides test kits for determiningthe absolute concentration or the relative concentration, or determiningthe presence or absence of the biomarkers of the present invention,thereby enabling the measurement of renal function and/or diagnosis ofrenal failure. The test kit can be used to diagnose the absence orpresence of renal failure/renal disease. The test kit can also be usedto predict the occurrence or to predict the stage of renal failure. Thetest kit can further be used to predict and/or monitor the success of atherapy for renal failure such as therapy by medications, therapy bydialysis, or therapy by kidney transplantation. Furthermore, the testkits of the present invention can be used for the pre-analysis of adiagnostic sample intended for other non-renal failure diagnosticprocedures. The test kits for pre-analysis determines whether renalfailure is present, as renal failure might impair other markers fornon-renal failure diseases or conditions, which are intended to bemeasured using the same sample.

The test kit can be used to diagnose and/or predict chronic renalfailure at a stage earlier than other diagnostics tests currentlyavailable (e.g. at Stage 2), such as creatinine clearance, Cystatin C.The test kit can also be used to early diagnose and/or predict acuterenal failure, such as at R-stage or I-stage.

The test kit can be used to analyze samples from a subject such as thosesamples described previously. The test can be done with pooled samplescollected during a certain period of time, e.g., after kidneytransplantation, during hemodialysis, or during diseases oftenassociated with renal failure such as sepsis. The test can be done withpooled samples of one or of more than one subject. The sample can beused directly, or after storage of the sample for various time periodsat various storage conditions, or the sample can be a pre-processed, forexample, the sample can be fractionated by chromatography, filtration,capillary electrophoresis, precipitation, liquid or other extractionmethods, and immunoprecipitation. The whole sample or subject or two ormore combined fractions of a pre-processed sample, originating from oneor more than one samples can be analyzed.

The test can be done once or several times over a period of time toanalyze the time course of qualitative or quantitative changes of abiomarker of the invention, e.g., the peptides of Seq ID NOs:1 and 2,the encoding nucleic acids, and fragments or modifications of thepeptides and nucleic acids.

Furthermore, the test kit may comprise binding agents for the antibodiesbinding specifically to the biomarkers of the invention, or antibodyfragments binding specifically to their antigen. The binding agents canbe present in the test kit in immobilized form, for example immobilizedto membranes, to microtiter plates, to mass spectrometric targets, toSELDI chips, to protein chips, to nucleic acid chips, to chromatographicresins, to magnetic particles, to metal particles, to agarose particles,or to other polymer particles. Alternatively, or in addition, thebinding agents can be present in the test kits in a form to facilitatetheir immobilization to surfaces and materials as noted above. Thebinding agents may be present in a labeled form, for example, labeledwith enzymes, fluorescent dyes, fluorescent proteins or proteinfragments or peptides, with different isotopes which can be used in massspectrometry and/or for radioactive measurements, with dyes measurableby chemiluminescence, photometry or by other measurement methods, withorganic or inorganic groups such as biotin, chitin, sugars, withlectins, with ligands for receptors or with ligands for otherstructures.

In a further embodiment, the kit of the present invention may optionallycomprise instructions for diagnosing, monitoring the progression orregression, or monitoring the treatment of the renal disorder. Theinstructions may be on how to use the test kit, how to prepare thesamples, what kind of samples to use, how to analyze and interpret theresults. The test kit may further comprise instructions on how to usethe kit for determining the presence or absence or prognosis of renalfailure and of distinct stages of renal failure, especially of chronicand/or acute renal failure.

EXAMPLES Example 1 Study Design and Probands

Healthy adult humans and humans suspected or known to suffer from renalfailure of different stages were enrolled in this study after theyprovided written informed consent. Laboratory and other parameters, suchas body weight, height, age, sex, creatinine in serum and/or urine, andprotein in serum and/or urine, were measured. If no 24 h urine samplewas available, the creatinine clearance was calculated by use of theCockcroft formula. Laboratory values determined were leukocytes,nitrite, pH, protein, glucose, ketone, urobilinogen, bilirubin, bloodand hemoglobin presence in urine measured using urine sticks. Not allmeasurements were performed on all subjects. In addition, the pastmedical history was considered.

The samples were subjected to a standardized protocol including additionof high concentrations of chaotropic salt and ultra filtration, followedby reverse-phase chromatography, followed by MALDI-mass spectrometry ofindividual fractions of the chromatography, followed by quantitative andqualitative analysis of the resulting data representing fraction,molecular mass and MALDI signal intensity for each detected substance.This procedure led to the identification of mass spectrometric signalssuitable to distinguish the different stages of renal failure.

Example 2 Sample Collection and Plasma Preparation

Blood samples were drawn from subjects by venipuncture from a cubitalvein, using a 20 gauche needle and a butterfly system with a maximumtubing length of 8 cm. If a tourniquet was applied, it did not remain inplace for longer than 1 min. As soon as the blood flowed into thecontainer the tourniquet was released partially. Prior to collecting ablood sample for subsequent plasma preparation, the blood samplingdevice was flushed with a few ml of the blood not used for plasmapreparation. Subsequently, blood was drawn into standard EDTA-containingsyringes (e.g., 9 ml EDTA-Monovette, Sarstedt, Nümbrecht, Germany).Within 30 min. of the blood drawing, the blood sample was centrifugedfor 10 min. at 2000×g at room temperature. After centrifugation, theplasma was transferred to a fresh tube and subjected to a secondcentrifugation for 15 min. at 2500×g at room temperature. The resulting,platelet-depleted plasma was transferred to a second fresh tube.Alternatively, aliquots of 1.5 mL plasma from the first centrifugationstep were filtered using a 10 mL syringe equipped with a celluloseacetate filter unit with a 0.2

M pore size and 5 cm² filtration area (e.g., Sartorius Minisart®,Sartorius, Göttingen, Germany). All plasma samples in this study wereprepared by the 2-step centrifugation method.

Within 30 min. of the preparation, the plasma samples were transferredto a −80° C. freezer and stored until further analyzed.

Example 3 Ultra Filtration of Plasma Samples in the Presence ofChaotropic Salt

After thawing in a water bath, the plasma samples were kept at roomtemperature until all proteins are re-dissolved. The sample (0.95 mL)was mixed with 3.92 mL of 8 M Guanidin hydrochloride solution, 0.08 mLpeptide standard. The pH was adjusted to pH 2-3 by the addition of 0.018mL of 30% [v/v] HCl. The diluted sample was centrifuged for 60 min. at4000×g at 23° C. in an ultra filtration device (Amicon Ulta-15, Art. No.UFC 905096, Millipore, Bedford, Mass.). Prior to use, the ultrafiltration devices were rinsed with water by adding 15 mL water into thedevice, centrifugation at 4000×g for 2 min. at 23° C., and discardingany water present in the device after this rinsing step. The finalvolume of 5.5 mL included 4.3 mL of the resulting filtrate (flow throughof the ultrafiltration) diluted with 1.2 mL of 0.1% Tri-Fluor-AceticAcid (TFA). This sample was stored at −80° C. until further analyzed.

Example 4 Reverse Phase (RP) Chromatography

The separation method carried out was a reverse phase (RP)chromatography. The separation of peptides and proteins was done using asource 5RPC, 4.6×150 mm reverse phase chromatography column (AmershamBiosciences Europe GmbH, Freiburg, Germany). Mobile phases of thefollowing compositions were used: mobile phase A: 0.06% (v/v)tri-fluor-acetic-acid, mobile phase B: 0.05% (v/v)tri-fluor-acetic-acid, 80% (v/v) acetonitrile. The chromatography tookplace at 33° C. using an HP 1100 with a micro flow cell, both suppliedby Agilent Technologies (Böblingen, Germany).

After thawing, samples prepared according to example 3 were centrifugedat 18000·g for 10 minutes. The resulting 0.75 mL plasma equivalent wasloaded onto the chromatography column. The chromatography conditionswere as follows: 5% mobile phase B at time 0 min, from time 1 to 45 min.continuous increase in the mobile phase B concentration to 50%, fromtime 45 to 49 min. continuous increase in the mobile phase Bconcentration to 100%, and subsequently up to time 53 min. constant 100%buffer B. Seven minutes after the start of the chromatography, 96fractions (0.5 ml each) were collected.

Example 5 Mass Spectrometric Analysis

For mass spectrometric analysis, typical positive ion spectra ofpeptides were produced in a MALDI-TOF mass spectrometer (matrix-assistedlaser desorption ionization). Suitable MALDI-TOF mass spectrometers,such as Voyager-DE, Voyager-DE PRO or Voyager-DE STR, are manufacturedby Applied Biosystems (Foster City, Calif.). BIFLEX manufactured byBruker Daltonik (Bremen, Germany) can also be used.

For the mass spectrometric analysis, the samples were prepared by mixingthem with a matrix substance that consists of an organic acid. Suitablematrix substances according to the invention are3,5-dimethoxy-4-hydroxycinnamic acid, alpha-cyano-4-hydroxycinnamic acidand 2,5-dihydroxybenzoic acid. A lyophilized equivalent obtained byreverse phase chromatography and corresponding to 0.015 mL plasma wasused to measure the peptides and/or proteins and/or standards. Thechromatographed sample was dissolved in 0.015 mL of a matrix solution.The matrix solution contains, for example, 10 g/Lalpha-cyano-4-hydroxycinnamic acid and 10 g/L L(−)fucose dissolved in asolvent mixture consisting of acetonitrile, water, trifluoroacetic acidand acetone in the ratio 49:49:1:1 by volume. After 0.0003 mL of thissolution was transferred to a MALDI carrier plate, the dried sample wasanalyzed in a Voyager-DE STR MALDI mass spectrometer.

The measurement took place in linear mode with delayed extractionTM. TheMALDI-TOF mass spectrometer can be employed to quantify peptides ifthese peptides are present in a concentration which is within thedynamic measurement range of the mass spectrometer, thus avoidingdetector saturation. There is a specific ratio between measured signaland concentration for each peptide, which means that the MALDI massspectrometry can preferably be used for the relative quantification ofpeptides. It is possible to measure the signal intensities of thestandards and of peptides originating from the sample.

Example 6 Data Analysis

Subsequent to fractionation as described in Example 4, each fraction wasindividually analyzed by MALDI mass spectrometry as described in Example5 resulting in 96 mass spectra for each sample. These 96 mass spectrawere electronically combined to a so called peptide display. The x-axisof these peptide displays depicts the molecular mass, the y-axis depictsthe fraction number and the color intensity represents the massspectrometric signal intensity.

Data pre-processing involved absolute scaling of MALDI profiles,baseline correction, and m/z-recalibration of the mass spectrometricdata. Then spectra were binned down to 1 Da resolution. Subsequent dataanalyses were performed using 4352 of these signals which had anintensity above 50 units in at least 20% of the samples. The signalintensities were stored using the chromatographic fraction and massspectrometric m/z ratio as labels. Considering redundancy due tomass/fraction shifts (e.g. the same peptide is present in 2, 3 or moreneighboring fractions), oxidation (the same peptide with and withoutoxidation is present in the sample, without the oxidation being presentin the sample at the time the sample was collected) and, double ortriple charged peptide forms (the same peptide being detected atmultiple positions due to various charge states of it resulting indifferent mass spectrometric signals of the same molecule), the numberof distinct peptides in the analysis was estimated to be about 1500.

Example 7 Mass Spectrometric Sequence Determination

The data of peptide displays were pre-processed by adjusting forbackground noise. Differences between peptide displays were calculatedby subtracting peptide displays from each other electronically.Detection of qualitative or quantitative differences between individualsamples or between groups of samples regarding substances present in thesamples, such as peptides or proteins, was done by comparison of themass spectrometric data, e.g. the signal intensities of thecorresponding substances. This was done using mass spectrometric signalintensities of individual samples and groups of samples, wherein thegroups were samples from healthy versus samples from subjects sufferingfrom different stages of kidney failure.

Peptides were identified using nanoSpray-MS/MS. This entailed a standardpeptide ion being selected in the mass spectrometer on the basis of itsspecific m/z (mass/charge) value in a manner known to the skilledworker. This selected ion was then fragmented by supplying collisionenergy with an collision gas, e.g. helium or nitrogen, and the resultingfragments of the standard peptide were detected in the mass spectrometerin an integrated analysis unit, and corresponding m/z values weredetermined (principle of tandem mass spectrometry). The fragmentationbehavior of peptides made unambiguous identification of the peptidespossible. In this specific case, the mass spectrometric analysis wasperformed using a Quadrupol-TOF Instrument, QStar-Pulsar model fromApplied Biosystems.

While the invention has been illustrated and described in the figuresand foregoing description, the same is to be considered as illustrativeand not restrictive in character, it being understood that only thepreferred embodiments have been shown and described and that all changesand modifications that come within the spirit of the invention aredesired to be protected. In addition, all references and patents citedherein are indicative of the level of skill in the art and herebyincorporated by reference in their entirety.

We claim:
 1. A peptide biomarker consisting of SEQ ID NO:
 2. 2. A kitfor diagnosing, monitoring the progression or monitoring the treatmentof a renal disorder, comprising: a. a peptide biomarker consisting ofSEQ ID NO: 2; and b. instructions to detect said biomarker in abiological sample.